1

WAT E R S SO LU T IO NS

ACQUITY UPLC H-Class System

Xevo TQD

ACQUITY UPLC BEH Column

ACQUITY UPLC Isolator Column

K E Y W O R D S

Phthalates, whisky, whiskey, brandy, gin,

tequila, distilled beverages, spirits, DEHP,

DBP, DINP, BBP, DMP, DNOP, DEP

A P P L I C AT IO N B E N E F I T S ■■ Separation and detection of 7 phthalates

in 11 minutes.

■■ Simple, quick “dilute and shoot” sample

preparation method for routine applications

where high sample throughput is required.

■■ Elimination of major phthalate contamination

using the ACQUITY UPLC® Isolator Column.

■■ Selective mass spectra obtained for all seven

phthalates with dominant precursor ion.

■■ Low limits of quantification achieved using

Waters® ACQUITY UPLC H-Class System

and Xevo® TQD.

■■ Single and robust method for a variety

of distilled beverages.

■■ Maintain consumer confidence and

ensure compliance in market.

IN T RO DU C T IO N

Phthalates, esters of phthalic acid, are often used as plasticizers for polymers such

as polyvinylchloride. They are widely applicable in various products including

personal care goods, cosmetics, paints, printing inks, detergents, coatings,

and food packaging. These phthalates have been found to leach readily into the

environment and food as they are not chemically bound to plastics. As such they

are known to be ubiquitously present in our environment.

Phthalates have been reported to show a variety of toxic effects related to

reproduction in animal studies, which has resulted in these compounds being

considered as endocrine disruptors. Screening food and beverages for phthalates

contamination is required by many legislative bodies, although regulations vary

from country to country in regards to acceptable daily tolerances and specific

migration limits.

Traditionally, phthalates have been analyzed by gas chromatography-mass

spectrometry (GC-MS), where derivatization and/or extraction and sample

preparation is often required to improve chromatographic separation.1 The

resulting GC-EI-MS spectra can lack selectivity, where the base ion, used for

identification and quantification of many common phthalates is the non-selective

m/z 149 (C8H5O3) ion. Furthermore, background contamination of phthalates

remains a significant challenge due to their ubiquitous presence.

In this application note phthalates were separated on a reversed-phase column

within 11 minutes using UltraPerformance Liquid Chromatography (UPLC®)

coupled to a tandem quadrupole mass spectrometer. In order to assess the

method’s applicability, various brands of distilled spirits were tested. Repeated

injections of the samples were made to evaluate method robustness over a

number of days. Limits of detection (LOD) and quantification (LOQ) of seven

key phthalates (DEHP, BBP, DBP, DNOP, DEP, DMP, and DINP) in the sample

will be presented.

A Simple, Fast, and Reliable LC-MS/MS Method for Determination and Quantification of Phthalates in Distilled BeveragesDimple Shah and Jennifer BurgessWaters Corporation, Milford, MA, USA

2

E X P E R IM E N TA L

Standards

Dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl

phthalate (DBP), benzyl butyl phthalate (BBP), bis(2-ethylhexyl)

phthalate (DEHP), di-n-octyl phthalate (DNOP), and diiso-nonyl

phthalate (DINP) were purchased from Sigma-Aldrich. The

stock solutions for each phthalate were prepared at 1 mg/mL

in methanol.

Samples

The samples analyzed in this application note consisted of an

assortment of various distilled beverages purchased in the

U.S.A. The sample description for each of the six samples is

listed in Table 1.

Sample name Type

1. Sample A Brandy (Brand A)

2. Sample B Gin

3. Sample C Whisky (Brand A)

4. Sample D Brandy (Brand B)

5. Sample E Tequila

6. Sample F Whisky (Brand B)

Table 1. List of distilled beverages.

Sample preparation

The distilled beverage samples were diluted 1:1 with water and

placed in LCGC Certified Glass 12 x 32 mm Screw Neck Total

Recovery Vials (p/n 186000384C) for LC-MS/MS analysis.

For the matrix matched spiked calibration, Sample F was spiked

with seven phthalates at 5, 10, 20, 50, and 100 ppb (µg/L)

and diluted 1:1 with water. It is important to note that the water

used for sample preparation should be screened for phthalates.

Any background contamination from the water will add to any

response in the sample and must be accounted for.

A Simple, Fast, and Reliable LC-MS/MS Method for Determination and Quantification of Phthalates in Distilled Beverages

LC conditions LC system: ACQUITY UPLC H-Class

Mobile phase A: Water + 0.1% formic acid

Mobile phase B: Methanol + 0.1% formic acid

Column: ACQUITY UPLC BEH C18 2.1 x 50 mm, 1.7 µm

Column temp.: 40 °C

Injection volume: 10 µL

Flow rate: 0.5 mL/min

Total run time: 11.0 min

Column: ACQUITY UPLC Isolator, 2.1 x 50 mm (p/n 186004476)

Needle wash: 90% Methanol + 0.1 % formic acid

Purge solvent: 10% Methanol

Seal wash: 10% Methanol

Time (min) %A %B Curve

Initial 60 40 6

0.5 60 40 6

5.0 1 99 6

8.0 1 99 6

8.5 60 40 6

11.0 60 40 6

MS conditionsMS System: Xevo TQD

Ionization mode: ESI +

Capillary voltage: 0.5 kV

Source temp.: 150 °C

Desolvation temp.: 500 °C

Desolvation gas: 1000 L/hr

Acquisition: Multiple Reaction Monitoring (MRM)

3

Data acquisition and processing

The data was acquired using MassLynx® Software, and processed using TargetLynx™ Application Manager.

For all compounds, two MRM transitions were acquired for quantification and confirmation purposes. The MRM

transitions, cone voltages, and collision energies for all compounds, along with expected retention times are

shown in Table 2.

NameRetention

time (min.)

Parent mass m/z

Cone voltage

(V)

Daughter 1 m/z

Collision energy 1

(V)

Daughter 2 m/z

Collision energy 2

(v)

DMP 1.37 195 15 133.0 20 163.0 10

DEP 3.14 223.1 20 149.1 20 177.1 10

DBP 5.01 279.1 15 149.0 15 208.0 5

BBP 4.97 313.2 10 149.0 10 205.1 5

DEHP 6.50 391.3 25 113.1 10 167.0 15

DNOP 6.62 391.3 30 149.0 15 261.2 10

DINP 6.73 419.4 35 127.1 35 149.0 20

Table 2. List of phthalates with MRM transitions and retention times.

R E SU LT S A N D D IS C U S S IO N

The chemical formula, mass (M+H+), and structure of all phthalates are shown in Table 3. The separation of the

seven phthalates was achieved using the ACQUITY UPLC H-Class System in 11 minutes. The retention times of

all phthalates are shown in Table 2.

Name CAS No. Chemical Formula

m/z (M+H+) Structure

Benzylbutyl phthalate (BBP)

85-68-7 C19H20O4 313.2

Dibutyl phthalate (DBP) 84-74-2 C16H22O4 279.1

Bis (2-ethylhexyl) phthalate (DEHP)

117-81-7 C24H38O4 391.3

Dibutyl phthalate (DMP) 131-11-3 C10H10O4 195.0

Diethyl phthalate (DEP) 84-66-2 C12H14O4 223.0

DI-n-octyl phthalate (DNOP)

117-84-0 C24H38O4 391.3

Di iso-nonyl phthalate (DINP)

28553-12-0 C26H42O4 419.4

Table 3. Chemical formula, mass, and structure of all seven phthalates that were studied.

A Simple, Fast, and Reliable LC-MS/MS Method for Determination and Quantification of Phthalates in Distilled Beverages

4

Figure 1. The ACQUITY UPLC Isolator Column separates BBP and background phthalates in the solvent standard.

Due to the ubiquitous contamination of phthalates in the environment, the background response from

phthalates can interfere during sample analysis. In order to remove this background interference, an

ACQUITY UPLC Isolator Column (p/n 186004476) with an extension tube that was placed in the flow path

between the mobile phase mixer and the sample manager injector. Background phthalates are retained on

the Isolator Column until the gradient elutes them through the analytical column. In this way, the phthalates

from the sample elute earlier than the background phthalates. Hence the Isolator Column separates any

background phthalate response from the phthalates in the sample to be analyzed. In addition, the peak shape

of the phthalates from the background is much broader than those coming from the sample. Figure 1 shows

an example of the separation of background phthalate contamination from the analyte of interest (BBP)

in a solvent standard.

Figure 2 shows a chromatogram of each of the

phthalates spiked in whisky sample F at 100 ppb

(µg/L). All of the phthalates including the pair of

isomers (DEHP and DNOP) were easily separated in

less than 10 minutes. As discussed previously, the

smaller background peaks in the chromatograms

of DEP, DEHP/DNOP, and BBP are background

contamination separated from the analytes of

interest using the ACQUITY UPLC Isolator Column.

DEHP DNOP

DINP

BBP

DBP

DEP

DMP

Figure 2. Separation of all the seven phthalates tested at 100 ppb (µg/L) in sample F.

A Simple, Fast, and Reliable LC-MS/MS Method for Determination and Quantification of Phthalates in Distilled Beverages

5

Linearity

Sample F (whisky brand B) was chosen for the matrix

spiked calibration curve. Sample F was pre-spiked

with the seven phthalates at concentrations of

5, 10, 20, 50, and 100 ppb, then diluted 1:1 prior

to analysis. Figure 3 shows the matrix spiked

calibration curve for all of the phthalates in sample

F. The co-efficient of determination (r2) of each

calibration curve was >0.99 for all phthalates in

sample F (Figure 3), and also in a solvent calibration

curve (data not shown).

Limit of detection (LOD) and quantification (LOQ):

All phthalates were spiked into sample F at various

levels (1 to 10 ppb) to determine LOD and LOQ. With

the exception of DINP, all phthalates were easily

detected at 1 ppb with a signal-to-noise (S/N) ratio

>10. For DINP, the LOD and LOQ limits were based on

the matrix spiked calibration standard that provided

a signal-to-noise ratio >3 and >10, respectively.

These are shown in Table 4 for sample F.

Figure 3. Matrix spiked calibration curve of all phthalates from 5 ppb (µg/L) to 100 ppb (µg/L) in sample F.

Compound name: DEHPCorrelation coefficient: r = 0.995267, r2 = 0.990557Calibration curve: 435.265 * x + 1742.05Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

ppb-0 10 20 30 40 50 60 70 80 90 100

Res

pons

e

-0

10000

20000

30000

40000

Compound name: DNOPCorrelation coefficient: r = 0.998477, r2 = 0.996957Calibration curve: 763.204 * x + 168.408Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

ppb-0 10 20 30 40 50 60 70 80 90 100

Res

pons

e

-0

20000

40000

60000

Compound name: DINPCorrelation coefficient: r = 0.995208, r2 = 0.990439Calibration curve: 95.9474 * x + -122.963Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

ppb-0 10 20 30 40 50 60 70 80 90 100

Res

pons

e

-0

2000

4000

6000

8000

10000

DINP

DNOPDEHP

Compound name: DMPCorrelation coefficient: r = 0.997256, r2 = 0.994520Calibration curve: 2819.84 * x + 19543.6Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

ppb-0 10 20 30 40 50 60 70 80 90 100

Res

pons

e

-0

100000

200000

300000

Compound name: DEPCorrelation coefficient: r = 0.996397, r2 = 0.992807Calibration curve: 2474.49 * x + 10264.5Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

ppb-0 10 20 30 40 50 60 70 80 90 100

Res

pons

e

-0

50000

100000

150000

200000

Compound name: DBPCorrelation coefficient: r = 0.995720, r2 = 0.991458Calibration curve: 4491.98 * x + 16161.4Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

ppb-0 10 20 30 40 50 60 70 80 90 100

Res

pons

e

-0

100000

200000

300000

Compound name: BBPCorrelation coefficient: r = 0.997755, r2 = 0.995515Calibration curve: 839.103 * x + 1264.79Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

ppb-0 10 20 30 40 50 60 70 80 90 100

Res

pons

e

-0

20000

40000

60000

80000

BBPDBP

DEPDMP

Name LOD LOQ

DMP 1 ppb 1 ppb

DEP 1 ppb 1 ppb

DBP 1 ppb 1 ppb

BBP 1 ppb 1 ppb

DEHP 1 ppb 1 ppb

DNOP 1 ppb 1 ppb

DINP 5 ppb 10 ppb

Table 4. LOD and LOQ of all phthalates in sample F.

A Simple, Fast, and Reliable LC-MS/MS Method for Determination and Quantification of Phthalates in Distilled Beverages

6

Figure 4. Chromatograms of BBP in brandy A, brandy B, gin, whisky, and tequila samples: spiked at 100 ppb (µg/L) and blank.

Figure 5. Chromatogram of DEHP in a solvent standard and brandy (sample A). Low level detection of DEHP in sample A at <5 ppb.

To study the applicability of the method to other distilled beverage types, all distilled beverage samples

listed in Table 1 were spiked with the seven phthalates at 100 ppb (µg/L) and analyzed. All phthalates were

successfully detected in all of the samples. Figure 4 shows the MRM chromatograms of BBP for each of the

samples. BBP was successfully detected in all of the distilled beverage samples at 100 ppb.

Standard : 100 ppb

Sample A : Brandy

6.50

6.50

Brandy: sample A 4.99

Spiked at 100 ppb

Blank

Gin: sample B 4.99

Spiked at 100 ppb

Blank

Tequila: sample E 4.99

Spiked at 100 ppb

Blank

4.99

Spiked at 100 ppb

Blank

Brandy: sample D

Whisky: sample C 4.99

Spiked at 100 ppb

Blank

Sample analysis

All of the alcoholic beverages (samples A to F) were

analyzed in triplicate. For the majority of samples,

no phthalates were detected. There was a trace level

of DEHP detected in brandy A (<5 ppb). Figure 5

shows a chromatogram of DEHP (2 MRM transitions)

in a solvent standard and in brandy A.

A Simple, Fast, and Reliable LC-MS/MS Method for Determination and Quantification of Phthalates in Distilled Beverages

7

Robustness study

To assess the repeatability and robustness of the method, 100 injections of sample F spiked at 100 ppb were

performed. Figure 6 shows an example TrendPlot of DMP over these 100 injections. The %RSD (retention time

and area count) and relative precision (CV) for all compounds are shown in Table 5. The %RSDs for area count

and retention time were <6% and 0.1% respectively for all compounds over the 100 injections.

Figure 6. TrendPlot of DMP for 100 injections.

Table 5. Percentage relative standard deviation (retention time and area) and CV (area) for all phthalates (n= 100 injections).

Name RSD % (RT)

RSD % (area)

CV (area)

DMP 0.096 4.55 0.04

DEP 0.092 1.87 0.01

DBP 0.050 2.05 0.02

BBP 0.059 1.95 0.01

DEHP 0.035 3.82 0.03

DNOP 0.053 5.90 0.05

DINP 0.067 4.98 0.04

A Simple, Fast, and Reliable LC-MS/MS Method for Determination and Quantification of Phthalates in Distilled Beverages

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

Waters, ACQUITY UPLC, Xevo, UPLC, MassLynx, and The Science of What’s Possible are registered trademarks of Waters Corporation. TargetLynx is a trademark of Waters Corporation. All other trademarks are the property of their respective owners

©2015 Waters Corporation. Produced in the U.S.A. May 2015 720005403EN AG-PDF

CO N C LU S IO NS■■ A simple and quick “dilute and shoot” method has been developed for accurate

quantification of seven phthalates.

■■ The phthalates of interest were easily separated from background

contamination using the ACQUITY UPLC Isolator Column.

■■ Excellent robustness, linearity, and levels of detection <10 ppb for all of the

phthalates were achieved using the ACQUITY UPLC H-Class System coupled

with Xevo TQD.

Acknowledgment The authors would like to acknowledge Robert Fotheringham and Edward Hayward

at Chivas Brothers, Glen Keith Technical Centre, Glen Keith Distillery, Station

Road, Keith, Banffshire, AB55 5BU, Scotland for their valuable input to this

application.

Reference

1. H Y Shen. Simultaneous screening and determination eight phthalates in plastic products for food use by sonication-assisted extraction/GC-MS methods. Elsevier. 2005: 734–739.